In cryogenic processes for the preservation of biological substances such as blood, blood components, cell suspensions and cell tissues, the major problem resides in avoiding irreversible cell damage which can result during the freezing process and the subsequent thawing process, or the minimizing of such damage.
It has been proposed heretofore to limit the cell damage of biological substances of the character described by the addition of a cryophylactic protective additive or agent which serves to protect the cells against the effects of freezing and thawing and which is mixed with the cell suspension or other biological substance. Such protective agents increase the survival rate of the frozen cell materials.
Protective additives such as glycerin have been used heretofore, especially for the protection of blood against the effects of the deep-freezing process, and must be washed from the preserved biological substances after thawing because they can adversely affect the human organism. Considerable research has gone into the development of biologically innocuous protective additives and, when such are employed, the survival rate can be increased.
Investigations have shown that an important factor in avoiding the decomposition or destruction of the cells is the temperature gradient with which the cells are frozen. In other words, there are predeterminable cell-specific time-dependent temperature gradients at which cellular material, i.e., the biological substances described above, can be frozen to obtain a survival rate of about 98%. This latter percentage has been found to be a reasonable level for most cryogenic deep-freezing processes and, when reference is made herein to time-dependent cell-specific temperature gradients, it will be understood that such gradients are intended as will ensure a cell survival rate of about 98% following deep-freezing and thawing.
When the speed of the freezing process lies beneath this temperature gradient, the concentration of the extracellular liquid is increased during the freezing process by the freezing out of water therefrom. This results in an increase in the osmotic pressure between the inner-cell and outer-cell media. Furthermore, during the freezing process water is withdrawn from the cells themselves and this results in a concentration increase in the intracellular solution as well. This can give rise to denaturation of the proteins in the cell interiors. While the effects of such processes can be minimized by an increase in the speed of the freezing process, there nevertheless is a tendency at both excessively high speeds and low speeds to produce intercellular ice which, in any case, breaks down the cell walls and membranes.
Of course, the amount and type of protective agent will also influence the desired temperature gradient of the freezing process. For example, when mixtures of erythrocytes with glycerin in high concentrations of about 50% are subjected to deep-freezing at a temperature gradient of about 8 K/min (8.degree. Kelvin or Centigrade per minute), high survival rates of the blood cells are noted. For unprotected erythrocytes, the optimum temperature gradient is about 5000 K/min and even at this optimum, the maximum survival rate of the cells is found to be only about 60%.
Known processes for the deep-cooling preservation of biological substances, which can be contained in so-called bioreceptacles, either maintain the biological receptacle in a liquid nitrogen bath for a predetermined time period, sometimes with shaking in order to ensure effective mixture of the biological substance with the protective agent, or spray the bioreceptacle with liquid nitrogen while monitoring the temperature within the interior of the receptacle.
The receptacle which can be used in the prior-art systems and in the invention described below can be any synthetic-resin sac or other container conventionally used to receive mixtures of blood and protective agents or other biological substances admixed with protective agents.
By the technique described above, the freezing process cannot be accurately maintained at a predetermined cell-specific temperature gradient.
The immersion process, which can be limited only as to time, does not permit variation in the temperature gradient under such controls as to maintain a predetermined cell-specific temperature gradient and the optimum temperature gradient for any specific cell can, at best, only be approached.
The spray process permits a monitoring of the change of temperature with time by means of a thermoelement in the interior of the bioreceptacle, but has the disadvantage that there is a large time lag in the control process, i.e., the reaction time between a change in the supply of the coolant and the resulting change in the temperature in the interior of the bioreceptacle is considerable. This, too, prevents an accurate control of the temperature gradient.